The discovery of major aggregated proteins in neurodegenerative diseases provides the first insight into disease pathogenesis. Recently, the dominant protein component within ubiquitin positive inclusions of frontotemporal lobar degeneration (FTLD-U) and sporadic amyotrophic lateral sclerosis (ALS) was found to be TAR DNA-binding protein 43 (TDP-43). The common pathology observed in these diseases suggests parallel approaches to treatment based on the understanding of TDP-43 aggregation events. In normal cells TDP-43 is a nuclear RNA binding protein involved transcriptional regulation. However, pathologic TDP-43 has been reported to redistribute from the nucleus to the cytoplasm where it is aggregated, phosphorylated, ubiquitinated and/or cleaved. Our preliminary results show that over-expressed human TDP-43 and the alternative splicing isoform (TDP-S6) are phosphorylated, ubiquitinated and cleaved in mammalian cell cultures. The expressed TDP-43 is localized diffusely and almost exclusively in the nucleus. In contrast, the TDP-S6 isoform is predominantly aggregated in the cytoplasm, recapitulating the disease-specific pathological hallmark. Therefore, our hypothesis is that TDP-43 aggregation and toxicity depends on the specific production of spliced and/or posttranslationally modified TDP-43 isoforms (i.e. cleavage, phosphorylation and ubiquitination). To test this hypothesis,we will investigate the expression of spliced TDP-43 isoforms in normal brain and FTLD-U tissue using RT-PCR and quantitative mass spectrometry (MS) approaches. We will also use MS to characterize phosphorylated and cleaved TDP-43 isoforms. Finally, we will assess TDP-43 mediated aggregation and neurotoxicity by expressing full-length TDP-43 and the S6 isoform in cell lines and primary neuronal cultures. This study will provide the first detailed characterization of TDP-43 isoforms either isolated from FTLD-U brain tissue or expressed in culture. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are severe neurodegenerative disorders that together affect approximately 30,000 people in the United States. The study of TDP-43 will provide deeper understanding into the pathogenesis of these diseases and potentially lead to novel therapeutic strategies.